The human T-box mesodermal transcription factor Brachyury is a candidate target for T-cell mediated cancer immunotherapy. Identification of tumor antigens is essential in advancing immune-based therapeutic interventions in cancer. Particularly attractive targets are those molecules that are selectively expressed by malignant cells and that are also essential for tumor progression. We used a computer-based differential display analysis tool for mining of expressed sequence tag clusters in the human Unigene database and identified Brachyury as a novel tumor antigen. Brachyury, a member of the T-box transcription factor family, is a key player in mesoderm specification during embryonic development. Moreover, transcription factors that control mesoderm have been implicated in the epithelial-mesenchymal transition (EMT), which has been postulated to be a key step during tumor progression to metastasis. Reverse transcription-PCR analysis validated the in silico predictions and showed Brachyury expression in tumors of the small intestine, stomach, kidney, bladder, uterus, ovary, and testis, as well as in cell lines derived from lung, colon, and prostate carcinomas, but not in the vast majority of the normal tissues tested. An HLA-A0201epitope of human Brachyury was identified that was able to expand T lymphocytes from blood of cancer patients and normal donors with the ability to lyse Brachyury-expressing tumor cells. To our knowledge, this is the first demonstration that (a) a T-box transcription factor and (b) a molecule implicated in mesodermal development, i.e., EMT, can be a potential target for human T-cell mediated cancer immunotherapy. The Interleukin-2 immunotoxin denileukin diftitox reduces regulatory T cells and enhances vaccine-mediated T-cell immunity. CD4+CD25+Foxp3+ T regulatory cells (Treg cells) have been implicated in the lack of effective antitumor immunity. Denileukin diftitox (DAB389IL-2), a fusion protein of interleukin 2 (IL-2) and diphtheria toxin, provides a means of targeting Treg cells. In this study, we examined (1) the effect of denileukin diftitox on the deletion of Treg cells in various lymphoid compartments and (2) the dose scheduling of denileukin diftitox in combination with a recombinant poxviral vaccine to enhance antigen-specific immune responses. Treg cells in spleen, peripheral blood, and bone marrow of normal C57BL/6 mice were variously reduced after a single intraperitoneal injection of denileukin diftitox; the reduction was evident within 24 hours and lasted approximately 10 days. Injection of denileukin diftitox 1 day before vaccination enhanced antigen-specific T-cell responses above levels induced by vaccination alone. These studies show for the first time in a murine model (1) the differential effects of denileukin diftitox on Treg cells in different cellular compartments, (2) the advantage of combining denileukin diftitox with a vaccine to enhance antigen-specific T-cell immune responses, (3) the lack of inhibition by denileukin diftitox of host immune responses directed against a live viral vector, and (4) the importance of dose scheduling of denileukin diftitox when used in combination with a vaccine.